CYLIC UNIAXIAL COMPRESSION OF CRANIAL SUTURES CAUSES SOLUBLE FACTOR RELEASE IN VITRO

Participants: J. M. Taboas, J. L. Dreier, A. Young, A. Fribley, W. Wang, S. A. Goldstein, S. R. Buchman, M. A. Ignelzi, Jr.

Keywords: craniosyntosis, cell culture, murine, mechanical force

Introduction

During craniofacial morphogenesis, cranial suture patency (remaining open) and fusion (closing) are under the regulation of varied biological and mechanical factors. The sutures are the soft tissue regions that separate the cranial bony plates, allowing the skull to expand during growth. Previous studies have implicated an abnormal mechanical environment in the pathogenesis of craniosynostosis (premature fusion of the skull bones at the sutures, 1.2 out of 3,000 live births). Nevertheless, the mechanisms by which mechanical force drives cellular, molecular, and genetic events affecting suture morphogenesis remains unclear. Therefore, the purpose of this study was to assess suture morphogenesis, on a phenotypic and genotypic level, in cyclically loaded and co-cultured post-natal day 21 (PN21) wildtype mouse calvaria explants containing the sagittal sutures (which do not normally fuse in mice) in vitro.

Methods

Mouse calvaria were excised and small coupons (4x12 mm²) were cut out containing the sagittal suture and neighboring parietal bone. Coupons were cultured in serum free media as described in Opperman, et al. (1995, 1996). Five experimental groups were used. Clamped loaded and clamped unloaded coupons were fastened into custom loading devices, which applied cyclic uniaxial compression using a trapezoidal load protocol of 0.3 g at 1 Hz for 30 minutes (20 % ramp, 20 % plateau, near 1.0% strain). Co-culture loaded and co-culture unloaded specimens were not physically clamped or loaded but were cultured in the presence of clamped loaded or clamped unloaded specimens. Unclamped unloaded specimens were cultured apart from clamped specimens. After two weeks, specimens were paraffin embedded or cryo-processed, sectioned, and H&E stained. A variety of stains are currently being investigated to identify fibrous tissue vs. new bone production, including but not limited to Modified Mallory's, Giemory's, Masson's Trichrome, Toluene Blue, and Elastin staining on decalcified sections. Pico Sirius Red and Villaneuva's (in LR White embedded specimens) are being applied to undecalcified sections. Also, SEM and polarized light microscopy have been attempted. Bone production has been identified by the histology appearance of osteoid under H&E staining (increased eosin uptake) coupled with the presence of osteoblast-like cells. Two slides (near specimen center) from all specimens were scored for % active surface (active = osteoid production) by digitizing each section on a slide, identifying the aggregate length of active bone surfaces facing the suture, and normalizing by the total length of the bone fronts facing the suture. Paraffin sections were also hybridized with DIG labeled probe for Osf2/Cbfa1 (@ 7 days) and Bone Sialoprotein (@ 7 & 14 days) while cryosections were stained for Alkaline Phosphatase activity (@ 14 days).

Results

Uniaxial compression led to a response spectrum in both loaded and co-cultured specimens. In clamped coupons, an increase in osteoid activity was noted compared to unloaded controls evidence through augmented stain intensity and/or fusion. However, some clamped specimens were torn during the experimental procedure. In co-culture specimens, new osteoid activity at the suture increased in proportion to stimulation of the clamped specimen. Unloaded co-cultured specimens had no increase, loaded co-culture specimens had increased osteoid production, while torn co-culture specimens had increased osteoid or fused. Fusion in coupons occurred in discrete foci along the length of the suture. After 7 days in culture, Osf2 mRNA expression increased in suture cells in loaded specimens when compared to their unloaded and unclamped controls. However this increase was greater in the co-culture specimens, where Osf2 expression markedly increased in the loaded co-culture specimens compared to their unloaded controls. With respect to BSP, we also found mRNA expression increased in suture cells and on bone surfaces of loaded specimens compared to unloaded and unclamped controls. However, unlike Osf2, the expression of BSP in this case was lower in the co-culture specimens compared to the clamped specimens. Regarding Alkaline Phosphatase, we found enzyme production increased at 14 days in clamped loaded and loaded co-culture specimens along the bone surfaces when compared to their respective controls.

Current Work

Five main avenues are currently being investigated. First, although instrumental in pointing out the spectrum of response, many specimens were damaged or torn during the experimental protocol. Motivated by this fact and an interest in the effects of different types of mechanical force on suture morphogenesis, we developed a protocol for hydrostatic pressure loading of calvaria in vitro. We have initially loaded, in our custom apparatus, calvaria explants containing parietal, coronal, and posterior frontal sutures. Using a graded scale that indicates levels of bone front proximity and fusion at the suture, we found hydrostatic pressure increased coronal suture fusion. Second, we are preparing to expand our molecular assays to include hybridization with probes for MTwist, Msx2, and FgfR-1,2,3. Third, we intend to perform differential display on clamped loaded specimens compared to unloaded unclamped controls. As a first step, we intend to blot homogenized specimen samples (clamped loaded, co-culture loaded, and unclamped unloaded) on commercially available membrane arrays. To date, we have been collecting the necessary number of specimens for these assays. Fourth, we are developing and in vitro assay using human marrow stromal cells that will allow us to investigate the osteogenic properties of the conditioned media. Initially, this assay will discern the production of mineralized nodules using Von Kossa staining. To this end, we have been collecting and freezing conditioned media. Finally, we are collaborating with Michael Morris in order to identify osteoid production and investigate changes in osteoid composition due to loading and disease states using Ramen spectroscopy.